Intracellular pH is an important regulator of a broad range of metabolic and physiological processes, and plays a central role in cell function, growth and development (See, e.g., Intracellular pH: Its Measurement, Regulation and Utilization in Cellular Functions, Liss, New York, 1982). The pH gradient between the mitochondrion and the cytosol is believed to play a critical role in mitochondrial function, and hence in cellular energy production (Mitchell, Science 206, 1148-1149; 1979).
Disturbances in cellular pH regulation occur with many pathological conditions, and have sometimes been used diagnostically. The first successful use of in vivo NMR spectroscopy to diagnose human disease, a case of McArdle's syndrome, was based on a measurement of intracellular pH and its response to exercise (Ross et al., N. Engl. J. Med. 304, 1338-1342; 1981; Lewis et al., J. Appl. Physiol. 59, 1991-1994; 1985). It has often been observed that the intracellular pH or the pH regulation of carcinogenically transformed cells differs significantly from that of non-transformed cells. For example, dramatic intracellular acidification in response to hyperglycemia has been observed in murine RIF-1 tumors (Evelhoch et al., PNAS USA 81: 6496-6500; 1984). It has been proposed that the relationship between malignancy and pH is due in part to increased acidic glycolysis in conjunction with a decreased or absent Pasteur effect--the so-called "Warburg effect" (see, e.g., Goldberg, et al., Clin. Chem. 39,2360-2374; 1993; Weinhouse, et al., Krebsforsch. 87, 115-126; 1976). Also, the inner cells of solid tumors often become hypoxic as a result of reduced blood flow, and therefore are subject to acidification due to increased glycolytic flux. Hence, the ability to determine pH in cells and organisms is of fundamental significance in a broad range of biological processes, and is useful for analyzing and diagnosing many pathological conditions.
The traditional methods for determining intracellular pH include microelectrode techniques, studies of the distribution of weak acids and bases, and analyses based on extracted metabolites (Cohen and R. A. Iles, CRC Critical Revs. in Clinical and Lab. Sci. 6, 101-143; 1975). Although microelectrode technology has improved considerably, thereby extending the range of such measurements to individual cells, these methods are cumbersome and subject to large errors. The introduction of fluorescent microscopy combined with fluorescent pH indicators, and in vivo NMR techniques, has widened the availability of such measurements to perfused organs, experimental animals, and humans.
Typical NMR methods of pH determination utilize endogenous phosphorylated molecules whose chemical shift is pH dependent (Cohen, et al., in Critical Revs. in Clin. Lab. Sciences 6, 101; 1975; Jacobson, L.; Cohen, J. S. "Intracellular pH Measurements by NMR Methods" In: Noninvasive Probes of Tissue Metabolism. Cohen, J. S., Ed. Wiley: New York, 1982; Moon, R. B.; Richards, J. H. J. Biol. Chem. 248, 7276-7278; 1973; Roberts, J. K. M.; Wade-Jardetzky, N.; Jardetzky, O. Biochemistry 20, 5389; 1981); however, this approach is limited by several factors including the low concentration of inorganic phosphate (the most useful pH indicator) in many cells, significant interference from extracellular phosphate present in blood, buffers, or perfusates, the overlap of the phosphate resonance with resonances from various phosphomonoesters, and the absence in some cell types of a suitable metabolite to serve as a chemical shift reference. Consequently, exogenous NMR probes of intracellular pH have been described and developed in the art. (Thoma, J. W.; Steiert, J. G., Crawford, R. L., Ugurbil, K. Biochem. Bioshys. Res. Commun. 1986, 138, 1106; DeFronzo, M.; Gillies, R. J. J. Biol. Chem. 262, 11032 (1987); Szwergold, B. S.; Brown, T. R.; Freed, J. J. Cell. Physiol. 138, 227 (1989); Taylor, J. S.; Deutsch, C. J.; McDonald, G. G.; Wilson, D. F. Anal. Biochem. 114, 415 (1981); Taylor, J. S.; Deutsch, C. J. Biophys. J. 43, 261 (1983); Deutsch, C. J.; Taylor, J. S. Ann. N.Y. Acad. Sci. 508, 33 (1987); Deutsch, C. J.; Taylor, J. S. Bioshys J. 55, 799 (1989); Metcalfs, J. C.; Hesketh, T. R.; Smith, G. A. Cell Calcium 6, 188 (1985); Beech, J. S.; Iles, R. A. Biochem. Soc. Trans. 15, 871 (1987)).
In principle, exogenous NMR probes can be optimized for pH measurement based on criteria including: (1) resonances should exhibit a large change in chemical shift with change in pH (.DELTA..delta./.DELTA.pH) and a pK close to the physiological mean; (2) high detection sensitivity; (3) indicators should be capable of being loaded into cells and should not readily leak out once they are loaded; (4) there should be minimal overlap between the resonances of the indicator and those of other endogenous metabolites; and (5) if the chemical shift is the parameter of interest, the indicator should contain an internal reference so that no additional reference is required. Based on the above criteria, the use of fluorinated indicators is particularly attractive since criteria (2) and (4) can be completely satisfied, and, due to the high chemical shift sensitivity of .sup.19 F, the first criterion can be satisfied as well with an appropriate design of the indicator.
Similarly, exogenous fluorescent probes for intracellular pH designed for optimal pH determination would have: (1) a pK close to the level being monitored; (2) a quantum yield and fluorescence intensity sufficiently high so that cellular autofluorescence does not interfere with the measurement; (3) a fluorescence spectrum which exhibits minimal overlap with the natural autofluorescence spectrum of the cell; (4) a shift in the fluorescence excitation and/or emission maximum upon titration so that dual wavelength measurement and imaging techniques can be used; (5) sufficient charge to reduce interaction with hydrophobic binding sites in the cell and to lead to negligible leakage on the time scale of the measurements; and (6) a structure which is not readily subject to metabolic transformation in the cell, other than de-esterification used for loading.
One useful fluorescent indicator for intracellular pH is the molecule bis(carboxyethyl)carboxyfluorescein (BCECF) (Rink et al., J. Cell Biol. 95: 189-196; 1982). Other commercially available indicators are described, for example, in Haugland, R. P.; Larison, K. D., Handbook of Fluorescent Probes and Research Chemicals, 5th ed. (1992-1994) Eugene Oreg., pp. 129-142. In general, the available fluorescent intracellular pH probes fail to adequately meet the above criteria; in particular, they suffer from pK values which are not well matched to the cytosol, they tend to leak significantly, and they tend to bind with hydrophobic sites on intracellular consitutents such as cellular proteins.